The present invention relates to reversing inhibition of tumor suppression.
The failure of normal function of the retinoblastoma tumor suppressor gene (RB) has been implicated as a contributing factor in a number of tumor types, including retinoblastomas and osteosarcomas, as well as lung, breast, and bladder carcinomas. (For reviews, see Goodrich et al., Biochim. Biophys. Acta., Vol. 1155, pp. 43–61, 1993; Zacksenhaus et al., Adv. Cancer. Res., Vol. 61, pp. 115–141, 1993; Sellers et al., J. Clin. Oncol., Vol. 15, pp. 3301–3312, 1997; Lohmann, D. R., Hum. Mutat., Vol. 14, pp. 283–288, 1999). A major role of RB is repression of the E2F family of DNA-binding transcriptional activators, which regulate the cell cycle through various genes required for S-phase entry. In resting cells, RB exists in the hypophosphorylated form that binds directly to E2F. (Reviewed in Weinberg, R. A., Cell, Vol. 81, pp. 323–330, 1995; Dyson, N., Genes Dev., Vol. 12, pp. 2245–2262, 1998). Importantly, mutations in E2F-recognition sequences, at least in some promoters, lead to derepression in G0/G1 cells, rather than repression in S-phase. (Neuman et al., Mol. Cell. Biol., Vol. 14, pp. 6607–6615, 1994). Although RB binds to the promoters only through E2F, RB is capable of repressing not only E2F, but also various activators that bind to E2F-responsive promoters. It has been proposed that chromatin modifiers, including histone deacetylases, (Brehm et al, Nature, Vol. 391, pp. 597–601, 1998), ATP-dependent chromatin remodeling factors (Zhang et al., Cell, Vol. 101, pp. 79, 2000), and DNA methyltransferases (Fuks et al., Nat. Genet., Vol. 24, pp. 88–91, 2000; Robertson et al., Nat. Genet., Vol. 25, pp. 338–3342, 2000) are involved in the mechanisms of this active repression. (Harbour et al., Curr. Opin. Cell Biol., Vol. 12, pp. 685–689, 2000).
Once RB becomes hyperphosphorylated, it dissociates from E2F resulting in expression of E2F-responsive genes. This hyperphosphorylation event at the time of the G1/S transition of the cell cycle, (For reviews, see Weinberg, Cell, Vol. 81, pp. 323–330, 1995; Sherr, “Cancer cell cycles”, Science, Vol. 274, pp. 1672–1677, 1996; Dyson, Genes Dev., Vol. 12, pp. 2245–2262, 1998; Mittnacht, Vol. 8, pp. 21–27, 1998) is thought to occur through the enzymatic activity of cyclin-dependent kinases (CDK). Accordingly, RB regulates S-phase entry through binding to E2F in a cell cycle-dependent manner. This cell cycle-dependent regulation is disturbed by viral transforming factors, including adenovirus E1A, simian virus 40 large-T antigen, and human papillomavirus (HPV) E7. (For reviews, see Zalvide et al., Mol. Cell. Biol., Vol. 15, pp. 5800–5810, 1995; Flint et al., Annu. Rev. Genet., Vol. 31, pp. 177–212, 1997). These transforming factors bind to the evolutionally conserved C-terminal region of RB, referred to as the pocket domain, and inhibit access of RB to E2F, leading to loss of G1 control.
In mammals, two proteins, namely p107 and p130, are structurally and functionally related to RB (For reviews, see Dyson, N., Genes Dev., Vol. 12, pp. 2245–2262, 1998; Lipinski et al., Oncogene, Vol. 18, pp. 7873–7882, 1999). All family members, namely RB, p107, and p130, bind to E2F and actively inhibit E2F-responsive transcription, leading to G0/G1 arrest. Although the RB family members are similar in these properties, they are differentially expressed during mouse development (Reviewed in Jiang et al., Oncogene, Vol. 14, pp. 1789–1797, 1997; Lipinski et al., Oncogene, Vol. 18, pp. 7873–7882, 1999). While RB nullzygous mutant embryos die at midgestation with multiple defects (Clarke et al., Nature, Vol. 359, pp. 328–330, 1992; Jacks et al., Nature, Vol. 359, pp. 295–300, 1992; Lee et al., Nature, Vol. 359, pp. 288–294, 1992), p107 and p130 nullzygous mice do not have any obvious developmental or tumor phenotype (Cobrinik et al, Genes Dev., Vol. 10, pp. 1633–1644, 1996; Lee et al., Genes Dev., Vol. 10, pp. 1621–1632, 1996). This phenotypic difference may be due to unique roles of RB and/or distinct expression profiles of RB. On the other hand, mouse embryonic fibroblasts carrying inactivating disruptions in all three RB gene family members are viable and proliferate in culture (Dannenberg et al., Genes Dev., Vol 14, pp. 3051–3064, 2000; Sage et al., Genes Dev, Vol. 14, pp. 3037–3050, 2000). Importantly, triple knockout fibroblasts have a shorter cell cycle and are insensitive to G0/G1 arrest signals following contact inhibition or serum starvation. These results support the view that the RB family members play an essential role in growth arrest.
In mammals, the E2F family has six members, namely E2F-1 to -6 (For reviews, see Dyson, N., Genes Dev., Vol. 12, pp. 2245–2262, 1998; Black et al., Gene, Vol. 237, pp. 281–302, 1999). All family members recognize the same DNA sequence as a heterodimer with either DP-1 or DP-2. E2F-6 differs from other E2F family members in that it lacks the transactivation and RB-binding domains, suggesting that it acts antagonistically to other E2F family members by occupying the binding sites on promoters (For reviews, see Cartwright et al., Oncogene, Vol. 17, pp. 611–623, 1998; Gaubatz et al., Proc. Natl. Acad. Sci. U.S.A., Vol. 95, pp. 9190–9195, 1998; Trimarchi et al., Proc. Natl. Acad. Sci. U.S.A., Vol. 95, pp. 2850–2855, 1998). On the other hand, E2F-1 to -5 all have transactivation and RB-binding domains. While their function could be partly redundant, several lines of evidence indicate specific roles for each E2F member (Reviewed in Dyson, N., Genes Dev., Vol. 12, pp. 2245–2262, 1998; Black et al., Gene, Vol. 237, pp. 281–302, 1999). First, each E2F protein preferentially binds to different RB family members: RB binds to E2F-1 to -4; p107 interacts with E2F-4; and p130 interacts with E2F-4 and -5. In addition, E2F-1 to -5 are differently regulated according to cell type and developmental stage. Furthermore, E2F-1 to -3 appear to be exclusively nuclear, whereas a significant portion of E2F-4 and E2F-5 are present in cytoplasm (Allen et al., J. Cell. Sci., Vol. 110, pp. 2819–2831, 1997; Lindeman et al., Proc. Natl. Acad. Sci. U.S.A., Vol. 94, pp. 5095–5100, 1997; Verona et al., Mol. Cell. Biol., Vol. 17, pp. 7268–7282, 1997).
Although RB was identified over a decade ago as the first tumor suppressor (Friend et al., Nature, Vol. 323, pp. 643–646, 1986; Fung et al., Science, Vol. 236, pp. 1657–1661, 1987; Lee et al., Nature, Vol. 329, pp. 642–645, 1987), to our knowledge RB has never been purified to homogeneity. Here, we report its purification in a native form, and we demonstrate that RB is present in a complex. The 600 kDa subunit, referred to as microtubule-associated factor (MTAF) 600, interacts directly with RB and microtubules and plays a role in active repression of E2F-responsive genes, cell cycle arrest, and genomic stability. These findings indicate that RB functions as a complex in vivo.
Because of the importance of RB in tumor suppression and growth arrest, and the demonstrated occurrence of tumors in subjects in which the RB gene has been mutated, there is significant clinical interest in identifying how the process of tumor suppression can be manipulated. In the future, as prognostic tests for a variety of diseases improve, it will be desirable to modulate the expression of key proteins associated with disease.